1. Field of the Invention
The invention relates to a method for measuring the activity of cholesterol oxidase and an apparatus therefor, and also to a method for estimating the toxicity of chemical compounds and an apparatus therefor, and more particularly to such methods and apparatuses utilizing monolayers at the air-water interface.
2. Description of the Related Art
A prior method for measuring the activity of cholesterol oxidase is known as a method for biochemical analysis of cholesterol oxidase, utilized in a quantitative reaction of cholesterol which is one item of clinical examinations. Hereinbelow will be explained several prior methods for measuring the activity of cholesterol oxidase.
The first method is one reported in Methods of enzymatic analysis, Vol. 1, Fundamentals, 3rd edition, 1983. As shown in an equation (A), cholesterol is oxidized in the presence of cholesterol oxidase working as a catalyst, to thereby produce cholestenone and hydrogen peroxide (H.sub.2 O.sub.2). EQU Cholesterol.fwdarw.Cholestenone +H.sub.2 O.sub.2 (A)
This method measures the activity of cholesterol oxidase by directly measuring hydrogen peroxide produced in accordance with the equation (A) with an ultraviolet (UV) ray.
In the second method, methanol is first oxidized to formaldehyde with H.sub.2 O.sub.2 produced in accordance with the equation (A) and catalase as a catalyst. Then, the thus produced formaldehyde is reacted with acetylacetone and ammonia, and thus there is produced 3,5-diacetyl-1,4-dihydrolutidine. In this second method, the degree of activity of cholesterol oxidase can be measured by measuring the absorbance of 3,5-diacetyl-1,4-dihydrolutidine at the wavelength in the range from 405 nm to 415 nm.
In the third method, hydrogen peroxide (H.sub.2 O.sub.2) produced in accordance with the equation (A) is reacted with phenol and 4-aminoantipyrine in the presence of peroxidase. Then, a dye produced by such a reaction measured at the wavelength of 515 nm by means of absorptiometry to thereby measure the activity of cholesterol oxidase.
In the fourth method, hydrogen peroxide (H.sub.2 O.sub.2) produced in accordance with the equation (A) is reacted with MBTH (3-methyl-2-benzothiazolinone hydrazone) and dimethylaniline in the presence peroxidase. Then, a dye produced by such a reaction is measured at the wavelength of 600 nm by means of absorptiometry to thereby measure the activity of cholesterol oxidase.
In the fifth method, hydrogen peroxide (H.sub.2 O.sub.2) produced in accordance with the equation (A) is reacted with ABTS (2,2'-azino-di-[3-ethylbenzthiazolinesulphonic acid]) in the presence of peroxidase. Then, a cation radical produced by such a reaction is measured at the wavelength in the range from 420 nm to 436 nm to thereby measure the activity of cholesterol oxidase.
In the sixth method, luminol is oxidized by hydrogen peroxide (H.sub.2 O.sub.2) produced in accordance with the equation (A) in the presence of peroxidase. Then, the chemical luminescence energy generated in such oxidization is measured to thereby measure the activity of cholesterol oxidase.
It is illegal to directly test human beings to analyze the toxic effects of chemical compounds on the human body. Hence, conventional methods for estimating the toxicity of chemical compounds use mammals other than a human being, such as animal cells and micro-organisms, to test medicines, food additives, industrial wastes, organism producing products and so on as to whether they have general toxicity, such as acute toxicity, short term toxicity and long term toxicity, particular toxicity such as mutagen, partial stimulus, allergy, tumogenicity, teratogenicity and propagation, and residence in a human body concerning respiration, metabolism, accumulation and excretion. Furthermore, general biological estimates such as pharmacological estimates with respect to cell toxicity are made to thereby predict the toxicity of chemical compounds toward a human being. For instance, Japanese Patent Publication No. 5-74358, which is based on U.S. patent application Ser. No. 623,183, published on Oct. 18, 1994 in Japan has disclosed a method for determining the cell toxicity. In this method, while cells are being cultured, fluorescent substances and a drug are introduced to the cells. By analyzing the change of fluorescence in the cells, the sensitivity of the cells to the drug is measured.
Cholesterol is not soluble in water. Accordingly, in all conventional methods for measuring the activity of cholesterol oxidase, substrate cholesterol is dispersed in water by using a surfactant and then the activity of cholesterol oxidase is measured. However, the thus obtained enzymatic activity is not one between cholesterol and cholesterol oxidase, but one representing the interaction among cholesterol, surfactant and cholesterol oxidase. Accordingly, the activity of cholesterol oxidase is varied in dependence on a surfactant used. In addition, the above mentioned prior method in which a surfactant, enzyme and a dye have to be used requires complex handling for measurement.
The conventional methods for estimating the toxicity of chemical compounds mainly use an animal other than a human being and a cell for conducting a test. In the case of using an animal other than a human being as an object to be tested, it is required to spend a lot of cost, time and labor for breeding animals and for estimating the toxicity. Furthermore, an animal test is not preferable in view of the prevention of cruelty to animals. In the case of using cells, much time and labor is required for culturing cells, and it takes a lot of time to determine the estimate of toxicity because the handling for the estimate is quite complicated.